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Browsing by Author "Retamal, Mauricio"

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4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation
(2020) Retamal, Mauricio; Fiori, Mariana C.; Fernández-Olivares, Ainoa; Linsambarth, Sergio; Peña, Francisca; Quintana, Daisy; Stehberg, Jimmy; Altenberg, Guillermo A.
Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.
Are Polyunsaturated Fatty Acids Implicated in Histaminergic Dysregulation in Bipolar Disorder?: AN HYPOTHESIS
(2018) Riveros, María E.; Retamal, Mauricio
Bipolar disorder (BD) is an extremely disabling psychiatric disease, characterized by alternate states of mania (or hypomania) and depression with euthymic states in between. Currently, patients receive pharmacological treatment with mood stabilizers, antipsychotics, and antidepressants. Unfortunately, not all patients respond well to this type of treatment. Bipolar patients are also more prone to heart and metabolic diseases as well as a higher risk of suicide compared to the healthy population. For a correct brain function is indispensable a right protein and lipids (e.g., fatty acids) balance. In particular, the amount of fatty acids in the brain corresponds to a 50–70% of the dry weight. It has been reported that in speciﬁc brain regions of BD patients there is a reduction in the content of unsaturated n-3 fatty acids. Accordingly, a diet rich in n-3 fatty acids has beneﬁcial effects in BD patients, while their absence or high levels of saturated fatty acids in the diet are correlated to the risk of developing the disease. On the other hand, the histamine system is likely to be involved in the pathophysiology of several psychiatric diseases such as BD. Histamine is a neuromodulator involved in arousal, motivation, and energy balance; drugs acting on the histamine receptor H3 have shown potential as antidepressants and antipsychotics. The histaminergic system as other neurotransmission systems can be altered by fatty acid membrane composition. The purpose of this review is to explore how polyunsaturated fatty acids content alterations are related to the histaminergic system modulation and their impact in BD pathophysiology.
Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala
(2021) Linsambarth, Sergio; Carvajal, Francisco; Moraga, Rodrigo; Mendez, Luis; Tamburini, Giovanni; Jimenez, Ivanka; Verdugo, Daniel; Gómez, Gonzalo I; Jury, Nur; Martínez, Pablo; Van Zundert, Brigitte; Varela, Lorena; Retamal, Mauricio; Martin, Claire; Altenberg, Guillermo; Fiori, Mariana; Cerpa, Waldo; Orellana, Juan; Stehberg, Jimmy
Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.
ATP is required and advances cytokine-induced gap junction formation in microglia in vitro
(Hindawi Publishing Corp., 2013) Saez, Pablo; Shoji, Kenji; Retamal, Mauricio; Harcha, Paloma; Ramirez, Gigliola; Jiang, Jean; von Bernhardi, Rommy; Saez, Juan
Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs), affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-/IFN--induced dye coupling, probably through the induction of IL-1 release. Moreover, TNF-/IFN-, but not TNF- plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-/IFN-, but not TNF- plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-/IFN- in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.
Biphasic effect of linoleic acid on connexin 46 hemichannels
(2011) Retamal, Mauricio; Evangelista-Martínez, Flavio; León-Paravic, Carmen; Altenberg, Guillermo; Reuss, Luis
Connexins form hemichannels at undocked plasma membranes and gap-junction channels (GJCs) at intercellular contacting zones. Under physiological conditions, hemichannels have low open probabilities, but their activation under pathological conditions, such as ischemia, induces and/or accelerates cell death. Connexin 46 (Cx46) is a major connexin of the lens, and mutations of this connexin induce cataracts. Here, we report the effects of linoleic acid (LA) on the electrical properties of Cx46 GJCs and hemichannels expressed in Xenopus laevis oocytes. LA has a biphasic effect, increasing hemichannel current at 0.1 mu M and decreasing it at concentrations of 100 mu M or higher. The effects of extracellular and microinjected LA conjugated to coenzyme A (LA-CoA) suggest that the current activation site is accessible from the intracellular but not extracellular compartment, whereas the current inhibitory site is either located in a region of the hemichannel pore inaccessible to intracellular LA-CoA, or requires crossing of LA through an organelle membrane. Experiments with other fatty acids demonstrated that the block of hemichannels depends on the presence of a hydrogenated double bond at position 9 and is directly proportional to the number of double bonds. Experiments in paired oocytes expressing Cx46 showed that LA does not affect GJCs. The block by unsaturated fatty acids reported here opens the possibility that increases in the concentration of these lipids in the lens induce cataract formation by blocking Cx46 hemichannels.
Carbon monoxide (CO) is a novel inhibitor of connexin hemichannels
(The American Society for Biochemistry and Molecular Biology, 2014) León-Paravic, Carmen; Figueroa, Vania; Guzmán, Diego; Valderrama, Carlos; Vallejos, Antonio; Fiori, Mariana; Altenberg, Guillermo; Reuss, Luis; Retamal, Mauricio
Hemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or "free HCs" at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions.
Carbon Monoxide Modulates Connexin Function through a Lipid Peroxidation-Dependent Process: A Hypothesis.
(Lausanne : Frontiers Research Foundation, 2016) Retamal, Mauricio
Hemichannels are ion channels composed of six connexins (Cxs), and they have the peculiarity to be permeable not only to ions, but also to molecules such as ATP and glutamate. Under physiological conditions they present a low open probability, which is sufficient to enable them to participate in several physiological functions. However, massive and/or prolonged hemichannel opening induces or accelerates cell death. Therefore, the study of the molecular mechanisms that control hemichannel activity appears to be essential for understanding several physiological and pathological processes. Carbon monoxide (CO) is a gaseous transmitter that modulates many cellular processes, some of them through modulation of ion channel activity. CO exerts its biological actions through the activation of guanylate cyclase and/or inducing direct carbonylation of proline, threonine, lysine, and arginine. It is well accepted that guanylate cyclase dependent pathway and direct carbonylation, are not sensitive to reducing agents. However, it is important to point out that CO-through a lipid peroxide dependent process-can also induce a secondary carbonylation in cysteine groups, which is sensitive to reducing agents. Recently, in our laboratory we demonstrated that the application of CO donors to the bath solution inhibited Cx46 hemichannel currents in Xenopus laevis oocytes, a phenomenon that was fully reverted by reducing agents. Therefore, a plausible mechanism of CO-induced Cx46 hemichannel inhibition is through Cx46-lipid oxidation. In this work, I will present current evidence and some preliminary results that support the following hypothesis: Carbon monoxide inhibits Cx46 HCs through a lipid peroxidation-dependent process. The main goal of this paper is to broaden the scientific community interest in studying the relationship between CO-Fatty acids and hemichannels, which will pave the way to more research directed to the understanding of the molecular mechanism(s) that control the opening and closing of hemichannels in both physiological and pathological conditions.
Carbon monoxide: A new player in the redox regulation of connexin hemichannels
(International Union of Biochemistry and Molecular Biology, 2015) Retamal, Mauricio; León-Paravic, Carmen; Ezquer, Marcelo; Ezquer, Fernando; Del Río, Rodrigo; Pupo, Amaury; Martínez, Agustín; González, Carlos
Carbon monoxide (CO) is a gaseous transmitter that is known to be involved in several physiological processes, but surprisingly it is also becoming a promising molecule to treat several pathologies including stroke and cancer. CO can cross the plasma membrane and activate guanylate cyclase, increasing the cGMP concentration and activating some kinases, including PKG. The other mechanism of action involves induction of protein carbonylation. CO is known to directly and indirectly modulate the function of ion channels at the plasma membrane, which in turn have important repercussions in the cellular behavior. One group of these channels is hemichannels, which are formed by proteins known as connexins (Cxs). Hemichannel allows not only the flow of ions through their pore but also the release of molecules such as ATP and glutamate. Therefore, their modulation not only impacts cellular function but also cellular communication, having the capability to affect tissular behavior. Here, we review the most recent results regarding the effect of CO on Cx hemichannels and their possible repercussions on pathologies.
Cardiac remodeling and arrhythmogenesis are ameliorated by administration of Cx43 mimetic peptide Gap27 in heart failure rats
(2020) Lucero, Claudia M.; Andrade, David C.; Toledo, Camilo; Díaz, Hugo S.; Pereyra, Katherin V.; Diaz-Jara, Esteban; Schwarz, Karla G.; Marcus, Noah J.; Retamal, Mauricio; Quintanilla, Rodrigo A.; Del Rio, Rodrigo
Alterations in connexins and specifcally in 43 isoform (Cx43) in the heart have been associated with a high incidence of arrhythmogenesis and sudden death in several cardiac diseases. We propose to determine salutary efect of Cx43 mimetic peptide Gap27 in the progression of heart failure. Highoutput heart failure was induced by volume overload using the arterio-venous fstula model (AV-Shunt) in adult male rats. Four weeks after AV-Shunt surgery, the Cx43 mimetic peptide Gap27 or scrambled peptide, were administered via osmotic minipumps (AV-ShuntGap27 or AV-ShuntScr) for 4 weeks. Cardiac volumes, arrhythmias, function and remodeling were determined at 8 weeks after AV-Shunt surgeries. At 8th week, AV-ShuntGap27 showed a marked decrease in the progression of cardiac deterioration and showed a signifcant improvement in cardiac functions measured by intraventricular pressure-volume loops. Furthermore, AV-ShuntGap27 showed less cardiac arrhythmogenesis and cardiac hypertrophy index compared to AV-ShuntScr. Gap27 treatment results in no change Cx43 expression in the heart of AV-Shunt rats. Our results strongly suggest that Cx43 play a pivotal role in the progression of cardiac dysfunction and arrhythmogenesis in high-output heart failure; furthermore, support the use of Cx43 mimetic peptide Gap27 as an efective therapeutic tool to reduce the progression of cardiac dysfunction in high-output heart failure.
Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins
(American Society for Biochemistry and Molecular Biology by Sage, 2016) Pinto, Bernardo; García, Isaac; Pupo, Amaury; Retamal, Mauricio; Martínez, Agustín; Latorre, Ramón; González, Carlos
Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity.
Connexin and Pannexin hemichannels are regulated by redox potential
(Frontiers Research Foundation, 2014) Retamal, Mauricio
Connexins (Cxs) and Pannexins (Panxs) are two non-related protein families, having both the property to form hemichannels at the plasma membrane. There are 21 genes coding for different Cx based proteins and only 3 for Panx. Under physiological conditions, these hemichannels (Cxs and Panxs) present a low open probability, but when open, they allow the release of signaling molecules to the extracellular space. However, under pathological conditions, these hemichannels increase their open probability, inducing important lysis of metabolites, and ionic imbalance, which in turn induce the massive entry of Ca+2 to the cell. Actually, it is well recognized that Cxs and Panxs based channels play an important role in several diseases and -in many cases- this is associated with an aberrant hemichannel opening. Hemichannel opening and closing are controlled by a plethora of signaling including changes of the voltage plasma membrane, protein-protein interactions, and several posttranslational modifications, including protein cleavage, phosphorylation, glycosylation, hydroxylation and S-nitrosylation, among others. In particular, it has been recently shown that the cellular redox status modulates the opening/closing and permeability of at least Cx43, Cx46, and Panx1 hemichannels. Thus, for example, the gaseous transmitter nitric oxide (NO) can induce the S-nitrosylation of these proteins modulating in turn several of their properties. The reason is that the redox status of a cell is fundamental to set their response to the environment and also plays an important role in several pathologies. In this review, I will discuss how NO and other molecules associated with redox signaling modulate Cxs and Panx hemichannels properties.
Connexin and Pannexin-Based Channels in Oligodendrocytes: Implications in Brain Health and Disease
(2019) Vejar, Sebastián; Oyarzún, Juan; Retamal, Mauricio; Ortiz, Fernando; Orellana, Juan
Oligodendrocytes are the myelin forming cells in the central nervous system (CNS). In addition to this main physiological function, these cells play key roles by providing energy substrates to neurons as well as information required to sustain proper synaptic transmission and plasticity at the CNS. The latter requires a fine coordinated intercellular communication with neurons and other glial cell types, including astrocytes. In mammals, tissue synchronization is mainly mediated by connexins and pannexins, two protein families that underpin the communication among neighboring cells through the formation of different plasma membrane channels. At one end, gap junction channels (GJCs; which are exclusively formed by connexins in vertebrates) connect the cytoplasm of contacting cells allowing electrical and metabolic coupling. At the other end, hemichannels and pannexons (which are formed by connexins and pannexins, respectively) communicate the intra- and extracellular compartments, serving as diffusion pathways of ions and small molecules. Here, we briefly review the current knowledge about the expression and function of hemichannels, pannexons and GJCs in oligodendrocytes, as well as the evidence regarding the possible role of these channels in metabolic and synaptic functions at the CNS. In particular, we focus on oligodendrocyte-astrocyte coupling during axon metabolic support and its implications in brain health and disease.
Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing
(2021) Nardin, Chiara; Peres, Chiara; Putti, Sabrina; Orsini, Tiziana; Colussi, Claudia; Mazzarda, Flavia; Raspa, Marcello; Scavizzi, Ferdinando; Salvatore, Anna María; Chiani, Francesco; Tettey-Matey, Abraham; Kuang, Yuanyuan; Yang, Guang; Retamal, Mauricio; Mammano, Fabio
In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.
Connexin46 Expression Enhances Cancer Stem Cell and Epithelial-to-Mesenchymal Transition Characteristics of Human Breast Cancer MCF-7 Cells
(2021) Acuña, Rodrigo; Varas-Godoy, Manuel; Herrera-Sepúlveda, Diego; Retamal, Mauricio
Connexins (Cxs) are a family of proteins that form two different types of ion channels: hemichannels and gap junction channels. These channels participate in cellular communication, enabling them to share information and act as a synchronized syncytium. This cellular communication has been considered a strong tumor suppressor, but it is now recognized that some type of Cxs can be pro-tumorigenic. For example, Cx46 expression is increased in human breast cancer samples and correlates with cancer stem cell (CSC) characteristics in human glioma. Thus, we explored whether Cx46 and glioma cells, can set up CSC and epithelial-to-mesenchymal transition (EMT) properties in a breast cancer cell line. To this end, we transfected MCF-7 cells with Cx46 attached to a green fluorescent protein (Cx46GFP), and we determined how its expression orchestrates both the gene-expression and functional changes associated with CSC and EMT. We observed that Cx46GFP increased Sox2, Nanog, and OCT4 mRNA levels associated with a high capacity to form monoclonal colonies and tumorspheres. Similarly, Cx46GFP increased the mRNA levels of n-cadherin, Vimentin, Snail and Zeb1 to a higher migratory and invasive capacity. Furthermore, Cx46GFP transfected in MCF-7 cells induced the release of higher amounts of VEGF, which promoted angiogenesis in HUVEC cells. We demonstrated for the first time that Cx46 modulates CSC and EMT properties in breast cancer cells and thus could be relevant in the design of future cancer therapies
Connexins in melanoma: Potential role of Cx46 in its aggressiveness
(2020-11) Orellana, Viviana; Tittarelli, Andrés; Retamal, Mauricio
Melanoma is the most aggressive skin cancer, and in metastatic advanced states, it is completely refractory to chemotherapy. Therefore, it is relevant to understand the molecular bases that rule their aggressiveness. Connexins (Cxs) are proteins that under normal physiological conditions participate in intercellular communication, via the exchange of signaling molecules between the cytoplasm and extracellular milieu and the exchange of ions/second messengers between the cytoplasm of contacting cells. These proteins have shown important roles in cancer progression, chemo- and radiotherapy resistance, and metastasis. Accordingly, Cx26 and Cx43 seem to play important roles in melanoma progression and metastasis. On the other hand, Cx46 is typically expressed in the eye lens, where it seems to be associated with oxidative stress protection in fiber lens cells. However, in the last decade, Cx46 expression has been associated with breast and brain cancers, due to its role in potentiation of both extracellular vesicle release and cancer stem cell-like properties. In this review, we analyzed a potential role of Cx46 as a new biomarker and therapeutic target in melanoma.
Contribution of Connexin Hemichannels to the Decreases in Cell Viability Induced by Linoleic Acid in the Human Lens Epithelial Cells (HLE-B3)
(2019) Figueroa, Vania; Jara, Oscar; Oliva, Carolina; Ezquer, Marcelo; Ezquer, Fernando; Retamal, Mauricio; Martínez, Agustín; Altenberg, Guillermo; Vargas, Aníbal
Connexin (Cx) proteins form hemichannels that a allow bidirectional flow of ions and metabolites between the cytoplasm and extracellular space. Under physiological conditions, hemichannels have a very low probability of opening, but in certain pathologies, hemichannels activity can increase and induce and/or accelerate cell death. Several mechanisms control hemichannels activity, including phosphorylation and oxidation (i.e., S-nitrosylation). Recently, the effect of polyunsaturated fatty acids (PUFAs) such as linoleic acid (LA), were found to modulate Cxs. It has been seen that LA increase cell death in bovine and human lens cells. The lens is a structure allocated in the eye that highly depends on Cx for the metabolic coupling between its cells, a condition necessary for its transparency. Therefore, we hypothesized that LA induces lens cells death by modulating hemichannel activity. In this work, we characterized the effect of LA on hemichannel activity and survival of HLE-B3 cells (a human lens epithelial cell line). We found that HLE-B3 cells expresses Cx43, Cx46, and Cx50 and can form functional hemichannels in their plasma membrane. The extracellular exposure to 10–50 μM of LA increases hemichannels activity (dye uptake) in a concentration-dependent manner, which was reduced by Cx-channel blockers, such as the Cx-mimetic peptide Gap27 and TATGap19, La3+, carbenoxolone (CBX) and the Akt kinase inhibitor. Additionally, LA increases intracellular calcium, which is attenuated in the presence of TATGap19, a specific Cx43-hemichannel inhibitor. Finally, the long exposure of HLE-B3 cells to LA 20 and 50 μM, reduced cell viability, which was prevented by CBX. Moreover, LA increased the proportion of apoptotic HLE-B3 cells, effect that was prevented by the Cx-mimetic peptide TAT-Gap19 but not by Akt inhibitor. Altogether, these findings strongly suggest a contribution of hemichannels opening in the cell death induced by LA in HLE-B3 cells. These cells can be an excellent tool to develop pharmacological studies in vitro.
Contribution of peripheral and central chemoreceptors to sympatho-excitation in heart failure
(2017) Toledo, Camilo; Andrade, David C; Lucero, Claudia; Schultz, Harold D; Marcus, Noah; Retamal, Mauricio; Madrid, Carlos; Del Rio, Rodrigo
Chronic heart failure (CHF) is a major public health problem. Tonic hyper-activation of sympathetic neural outflow is commonly observed in patients with CHF. Importantly, sympatho-excitation in CHF exacerbates its progression and is strongly related to poor prognosis and high mortality risk. Increases in both peripheral and central chemoreflex drive are considered markers of the severity of CHF. The principal peripheral chemoreceptors are the carotid bodies (CBs) and alteration in their function has been described in CHF. Mainly, during CHF the CB chemosensitivity is enhanced leading to increases in ventilation and sympathetic outflow. In addition to peripheral control of breathing, central chemoreceptors (CCs) are considered a dominant mechanism in ventilatory regulation. Potentiation of the ventilatory and sympathetic drive in response to CC activation has been shown in patients with CHF as well as in animal models. Therefore, improving understanding of the contribution of the peripheral and central chemoreflexes to augmented sympathetic discharge in CHF could help in developing new therapeutic approaches intended to attenuate the progression of CHF. Accordingly, the main focus of this review is to discuss recent evidence that peripheral and central chemoreflex function are altered in CHF and that they contribute to autonomic imbalance and progression of CHF.
Cx46 hemichannel modulation by nitric oxide: Role of the fourth transmembrane helix cysteine and its possible involvement in cataract formation.
(2019) Retamal, Mauricio; Orellana, Viviana; Arévalo, Nicolás; Rojas, Cristóbal; Arjona, Rodolfo; Alcaíno, Constanza; González, Wendy; Canan, Jonathan; Moraga-Amaro, Rodrigo; Stehberg, Jimmy; Reuss, Luis; Altenberg, Guillermo
Under normal conditions, connexin (Cx) hemichannels have a low open probability, which can increase under pathological conditions. Since hemichannels are permeable to relatively large molecules, their exacerbated activity has been linked to cell damage. Cx46 is highly expressed in the lens and its mutations have been associated to cataract formation, but it is unknown whether Cx46 has a role in non-genetic cataract formation (i.e. aging and diabetes). Nitric oxide (NO) is a key element in non-genetic cataract formation and Cx46 hemichannels have been shown to be sensitive to NO. The molecular mechanisms of the effects of NO on Cx46 are unknown, but are likely to result from Cx46 S-nitrosation (also known as S-nitrosylation). In this work, we found that lens opacity was correlated with Cx46 S-nitrosation in an animal model of cataract. Consistent with this result, a NO donor increased Cx46 S-nitrosation and hemichannel opening in HLE-B3 cells (cell line derived from human lens epithelial cells). Mutagenesis studies point to the cysteine located in the fourth transmembrane helix (TM4; human C212, rat C218) as the NO sensor. Electrophysiological studies performed in Xenopus oocytes revealed that rat Cx46 hemichannels are sensitive to different NO donors, and that the presence of C218 is necessary to observe the NO donors' effects. Unexpectedly, gap junctions formed by Cx46 were insensitive to NO or the reducing agent dithiothreitol. We propose that increased hemichannel opening and/or changes in their electrophysiological properties of human Cx46 due to S-nitrosation of the cysteine in TM4 could be an important factor in cataract formation.
Cxs and Panx- hemichannels in peripheral and central chemosensing in mammals.
(Frontiers Research Foundation, 2014) Reyes, Edison; Cerpa, Verónica; Corvalán, Liliana; Retamal, Mauricio
Connexins (Cxs) and Pannexins (Panx) form hemichannels at the plasma membrane of animals. Despite their low open probability under physiological conditions, these hemichannels release signaling molecules (i.e., ATP, Glutamate, PGE2) to the extracellular space, thus subserving several important physiological processes. Oxygen and CO2 sensing are fundamental to the normal functioning of vertebrate organisms. Fluctuations in blood PO2, PCO2 and pH are sensed at the carotid bifurcations of adult mammals by glomus cells of the carotid bodies. Likewise, changes in pH and/or PCO2 of cerebrospinal fluid are sensed by central chemoreceptors, a group of specialized neurones distributed in the ventrolateral medulla (VLM), raphe nuclei, and some other brainstem areas. After many years of research, the molecular mechanisms involved in chemosensing process are not completely understood. This manuscript will review data regarding relationships between chemosensitive cells and the expression of channels formed by Cxs and Panx, with special emphasis on hemichannels.
Diseases associated with leaky hemichannels
(Frontiers Research Foundation, 2015) Retamal, Mauricio; Reyes, Edison; Garcia, Isaac; Pinto, Bernardo; Martinez, Agustin; Gonzalez, Carlos
Hemichannels (HCs) and gap junction channels (GJCs) formed by protein subunits called connexins (Cxs) are major pathways for intercellular communication. While HCs connect the intracellular compartment with the extracellular milieu, GJCs allow the interchange of molecules between cytoplasm of two contacting cells. Under physiological conditions, HCs are mostly closed, but they can open under certain stimuli allowing the release of autocrine and paracrine molecules. Moreover, some pathological conditions, like ischemia or other inflammation conditions, significantly increase HCs activity. In addition, some mutations in Cx genes associated with human diseases, such as deafness or cataracts, lead to the formation of more active HCs or "leaky HCs." In this article we will revise cellular and molecular mechanisms underlying the appearance of leaky HCs, and the consequences of their expression in different cellular systems and animal models, in seeking a common pattern or pathological mechanism of disease.

Browsing by Author "Retamal, Mauricio"

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